Methods and compositions for detection of specific nucleotide sequences

ABSTRACT

Methods and compositions are provided for the detection of specific nucleic acid sequences purified from cellular or tissue sources. More particularly, the present invention includes methods and compositions for the detection of nucleic acid sequences using a protection molecule that forms a protected nucleic acid sequence (PNAS) such as a triplex or duplex nucleic acid structure that includes the target nucleic acid sequence. An assay using the methods of the present invention may include one, two or three levels of specificity to minimize false positive signals. An assay using the methods or compositions of the present invention can be performed on large amounts of purified DNA in a single test, with high levels of sensitivity, thus eliminating the need for DNA amplification procedures.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 08/739,069,filed Oct. 26, 1996, now U.S. Pat. No. 5,962,225 which claims priorityto U.S. Provisional patent application Ser. No. 60/005,938, filed Oct.27, 1995, entitled Diagnostic Procedures and Process for Detection ofSpecific DNA Sequences. These applications are herein incorporated, intheir entirety, by reference.

TECHNICAL FIELD

The present invention comprises methods and compositions for detectingnucleic acid sequences. More particularly, the present inventioncomprises methods and compositions for detection of specific geneticsequences using nucleic acid target protection strategies. The methodsand compositions of the present invention can be used in the detectionof microorganisms, for diagnosis of infectious diseases in humans,animals and plants; assays of blood products, and for genetic analysisfor use in such areas as early detection of tumors, forensics, paternitydeterminations, transplantation of tissues or organs and genetic diseasedeterminations.

BACKGROUND OF THE INVENTION

Many target and signal amplification methods have been described in theliterature, but none are believed to offer the combination of highspecificity, simplicity, and speed. General reviews of these methodshave been prepared by Landegren, U., et al., Science 242:229-237 (1988)and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990). Thesemethods include polymerase chain reaction (PCR), PCR in situ, ligaseamplification reaction (LAR), ligase hybridization, Qβ bacteriophagereplicase, transcription-based amplification system (TAS), genomicamplification with transcript sequencing (GAWTS), nucleic acidsequence-based amplification (NASBA) and in situ hybridization. Some ofthese various techniques are described below.

Polymerase Chain Reaction (PCR)

PCR is the nucleic acid amplification method described in U.S. Pat. Nos.4,683,195 and 4,683,202 to Mullis. PCR consists of repeated cycles ofDNA polymerase generated primer extension reactions. The target DNA isheat denatured and two oligonucleotides, which bracket the targetsequence on opposite strands of the DNA to be amplified, are hybridized.These oligonucleotides become primers for use with DNA polymerase. TheDNA is copied by primer extension to make a second copy of both strands.By repeating the cycle of heat denaturation, primer hybridization andextension, the target DNA can be amplified a million fold or more inabout two to four hours. PCR is a molecular biology tool which must beused in conjunction with a detection technique to determine the resultsof amplification. The advantage of PCR is that it may increasesensitivity by amplifying the amount of target DNA by 1 million to 1billion fold in approximately 4 hours. The disadvantage is thatcontamination may cause false positive results, or reduced specificity.

Transcription-based Amplification System (TAS)

TAS utilizes RNA transcription to amplify a DNA or RNA target and isdescribed by Kwoh et al. (1989) Proc. Natl. Acad. Sci., USA 86:1173. TASuses two phases of amplification. In phase 1, a duplex cDNA is formedcontaining an overhanging, single-stranded T7 transcription promoter byhybridizing a polynucleotide to the target. The DNA is copied by reversetranscriptase into a duplex form. The duplex is heat denatured and aprimer is hybridized to the strand opposite that containing the T7region. Using this primer, reverse transcriptase is again added tocreate a double stranded cDNA, which now has a double stranded (active)T7 polymerase binding site. T7 RNA polymerase transcribes the duplex tocreate a large quantity of single-stranded RNA.

In phase 2, the primer is hybridized to the new RNA and again convertedto duplex cDNA. The duplex is heat denatured and the cycle is continuedas before. The advantage of TAS over PCR, in which two copies of thetarget are generated during each cycle, is that between 10 and 100copies of each target molecule are produced with each cycle. This meansthat 10⁶ fold amplification can be achieved in only 4 to 6 cycles.However, this number of amplification cycles requires approximatelythree to four hours for completion. The major disadvantage of TAS isthat it requires numerous steps involving the addition of enzymes andheat denaturation.

Transcriptions Amplification (3SR)

In a modification of TAS, known as 3SR, enzymatic degradation of the RNAof the RNA/DNA heteroduplex is used instead of heat denaturation, asdescribed by Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874.RNAse H and all other enzymes are added to the reaction and all stepsoccur at the same temperature and without further reagent additions.Following this process, amplifications of 10⁶ to 10⁹ have been achievedin one hour at 42° C.

Ligation Amplification (LAR/LAS)

Ligation amplification reaction or ligation amplification system usesDNA ligase and four oligonucleotides, two per target strand. Thistechnique is described by Wu, D. Y. and Wallace, R. B. (1989) Genomics4:560. The oligonucleotides hybridize to adjacent sequences on thetarget DNA and are joined by the ligase. The reaction is heat denaturedand the cycle repeated. LAR suffers from the fact that the ligases canjoin the oligonucleotides even when they are not hybridized to thetarget DNA. This results in a high background. In addition, LAR is notan efficient reaction and therefore requires approximately five hoursfor each cycle. Thus, the amplification requires several days forcompletion.

Qβ Replicase

In this technique, RNA replicase for the bacteriophage Qβ, whichreplicates single-stranded RNA, is used to amplify the target DNA, asdescribed by Lizardi et al. (1988) Bio/Technology 6:1197. First, thetarget DNA is hybridized to a primer including a T7 promoter and a Qβ 5'sequence region. Using this primer, reverse transcriptase generates acDNA connecting the primer to its 5' end in the process. These two stepsare similar to the TAS protocol. The resulting heteroduplex is heatdenatured. Next, a second primer containing a Qβ 3' sequence region isused to initiate a second round of cDNA synthesis. This results in adouble stranded DNA containing both 5' and 3' ends of the Qβbacteriophage as well as an active T7 RNA polymerase binding site. T7RNA polymerase then transcribes the double-stranded DNA into new RNA,which mimics the Qβ. After extensive washing to remove any unhybridizedprobe, the new RNA is eluted from the target and replicated by Qβreplicase. The latter reaction creates 10⁷ fold amplification inapproximately 20 minutes. Significant background may be formed due tominute amounts of probe RNA that is non-specifically retained during thereaction.

Chiron Signal Amplification

The Chiron system, as described by Urdea et al. (1987) Gene 61:253, isextremely complex. It utilizes 12 capture oligonucleotide probes, 36labeled oligonucleotides, 20 biotinylated immobilization probes that arecrosslinked to 20 more enzyme-labeled probes. This massive conglomerateis built-up in a stepwise fashion requiring numerous washing and reagentaddition steps. Amplification is limited because there is no cycle. Theprobes simply form a large network.

ImClone Signal Amplification

The ImClone technique utilizes a network concept similar to Chiron, butthe approach is completely different. The ImClone technique is describedin Kohlbert et al. (1989) Mol. and Cell Probes 3:59. ImClone first bindsa single-stranded M13 phage DNA containing targeted probe. To this boundcircular DNA is then hybridized about five additional DNA fragments thatonly bind to one end and the other end hangs freely out in the solution.Another probe set is then hybridized to the hanging portion of theprevious set of probes. The latter set is either labeled directly withan enzyme or it is biotinylated. If it is biotinylated, then detectionis via a streptavidin enzyme complex. In either case, detection isthrough an enzyme color reaction. Like the Chiron method, the ImClonemethod relies on build-up of a large network. Because there is norepeated cycle, the reaction is not geometrically expanded, resulting inlimited amplification.

While the nucleic acid amplification methods described above allow forthe detection of relatively small quantities of target nucleic acidmolecules, there is a need for the ability to detect target nucleic acidmolecules in a shorter amount of time with less background interference.Problems inherent in PCR and other amplification techniques involvesample contamination during the collection techniques and the presenceof amplicons (amplified target DNA). There are problems withnon-specific target amplification mediated by closely related sequencesand the production of primer dimers. There is also poor control ofspecificity, resulting in false positive reactions, and poor control ofsensitivity, resulting in false negative reactions. PCR results mustoften be confirmed and validated by other techniques such as probehybridization, Southern blotting or in situ hybridization.

Additionally, PCR and amplification techniques can only be used withvery small amounts of starting sample DNA, in the range of a maximum of1 microgram. This negates use of PCR techniques for the detection of lowcopy number nucleic acid targets. For example, early detection of HIVinfection, soon after the initial viral infection, would be almostimpossible to detect using PCR.

Thus, compositions, methods and kits are needed that are capable ofdetecting specific nucleic acid sequences and isolating them. Especiallyneeded are methods and kits that would allow for the detection of lowcopy number nucleic acid target sequences. Additionally, there is needfor methods and kits that provide the flexibility that would allow forisolation of nucleic acid sequences using a desired level ofspecificity.

What is also needed are methods that do not use amplificationtechniques, but do allow for the isolation of a specific target sequencefrom any amount of starting nucleic acid, especially large amounts, andhave the flexibility to accomplish the isolation at several levels ofspecificity, depending on the level of specificity desired.

SUMMARY OF THE PRESENT INVENTION

In accordance with the present invention, methods and compositions areprovided for the detection of specific nucleic acid sequences fromcellular or tissue sources. More particularly, the present inventionincludes methods and compositions for the detection of nucleic acidsequences using a protection molecule that forms a protected nucleicacid sequence (PNAS) such as a triplex or duplex nucleic acid structurethat includes the target nucleic acid sequence. The targent nucleic acidsequence is the specific sequence being detected. An assay using themethods of the present invention may include one, two or three levels ofspecificity to minimize false positive signals. An assay using themethods or compositions of the present invention can be performed onlarge amounts of purified DNA in a single test, with high levels ofsensitivity, thus eliminating the need for in vitro DNA amplificationprocedures.

When the target nucleic acid sequence is double-stranded, the structureformed with the protection molecule is a triplex. When the targetnucleic acid sequence is single-stranded, the structure formed with theprotection molecule is a duplex. In this disclosure, where triplexstructures are discussed, one can also substitute duplex structures orstructures using PNA (peptide-nucleic acid) and the appropriatenucleases. Assays using the methods of the present invention may bereferred to as TPA, Target Protection Assays.

The initial level of specificity utilizes protection molecules such asoligonucleotides or peptide-nucleic acids (PNA) to bind to specifictarget sequences of interest. Such binding may be accomplished byformation of Hoogstein-type hydrogen bonds. The protection molecule,bound to the target nucleic acid sequence, forms the protected nucleicacid sequence (PNAS). Once these PNAS structures are formed andstabilized in solution, the non-specific DNA is digested. For example,this digestion can be accomplished with a combination of endonucleasesand a double-strand-dependent exonuclease, such as DNA Exonuclease III(Exo III). The endonucleases used in this example are designed to cut onboth sides of the PNAS, leaving approximately 20 base pairs of DNA oneach side of the sequence. Exo III, an exonuclease which progressivelycleaves one strand of the DNA from the 3' end, is inhibited by thetriple helix structure. Using a combination of nucleases, theunprotected DNA sequences are digested completely. A method of thepresent invention involving a lower level of specificity would employ anaffinity molecule for capture and a reporter molecule for labeling inconjunction with the protection probe.

However, if a higher level of specificity is required, 5' flankingregions can be generated on either or both sides of the PNAS to allowfor assays employing two further levels of specificity. The structureformed, a PNAS with flanking regions is termed PNAS/tail. Following theselected digestion around the PNAS, a capture probe, such as anoligonucleotide complementary to one of the single stranded flankingregions, is added. The capture probe is allowed to hybridize to asingle-stranded region. For example, the capture probe could be anoligonucleotide that would bind to a single-stranded region and have anaffinity molecule attached. For example, the affinity molecule could bedidoxigenein or biotin. The capture probe comprises an affinity moleculeand is capable of associating with the PNAS.

A capturing system is used to isolate the PNAS with the capture probeattached. Any capturing system that is capable of binding to the captureprobe and separating the PNAS/tails with affinity molecule from themixture is contemplated. In the example used above, such a capturesystem may comprise using magnetic beads coated with anti-didoxigeneinantibodies for binding to the didoxigenein-capture probe portion or,streptavidin for binding to the biotin-capture probe portion. ThePNAS/tails with affinity molecule, now attached to the magnetic beads,are separated from non-specific complexes and washed to remove anynon-specific nucleic acid sequences. Such washing may use any washingtechnique known in the art. For example, a magnetic particle holdercould be used. Again, should this be the level of specificity required,the present invention comprises assays that also have a reportermolecule associated with the protection molecule or the capture probe.

A third level of specific detection involves the addition of a labeledreporter probe. The reporter probe comprises a detectable label and iscapable of associating with the PNAS. For example, the reporter probedmay comprise an oligonucleotide complementary to the 5' single-strandedtail that is part of the PNAS/tail. This 5' region may or may not be onthe opposite flanking tail to which the capture probe binds. Thereporter probe may be labelled with any labels known in the art such asradioactivity or non-radioactive labels such as labeled with biotin ordidoxigenein for indirect detection, or directly with a fluorescentreporter molecule, e.g., fluorescein, or chemiluminescent orbioluminescent labels. An excess of reporter probe is added to thewashed magnetic bead-triplex complex and allowed to hybridize. Detectionof the bound labelled reporter probe can be accomplished after washingby using detection devices specific for the type of label used. Forexample, if a fluorescent labeled reporter probe is used, the labeledsequences can be detected using a fluorometer or viewing the beadsthrough a fluorescent microscope. Alternatively, the amount of boundprobe can be directly assessed by fluorescent anisotropy with ananalyzer such as the Abbott TDM analyzer.

Compositions of the present invention include compositions comprisingthe components to practice the methods taught herein. For example, acomposition comprising a labeled protection molecule with an affinitymolecule could be used in an assay with a first level of specificity. Acomposition comprising a labeled protection molecule and a capture probecould be used in a level two specificity assay. A composition comprisinga protection molecule, a capture probe and a reporter probe could beused in a level three assay. It is to be understood that the individualmolecules, probes and components can also be provided individually.

The present invention is especially useful for detecting specificgenetic sequences. The present invention comprises methods such as theTarget Protection Assay (TPA) in all its formats, which have theadvantage of allowing the processing of very large amounts of purifiednucleic acids, thus eliminating the need for artificial amplificationprocedures such as PCR, while enabling the detection of a specifictarget sequence. In addition, the three levels of specificity--PNASformation, capture probe binding, and reporter probe binding--reducetechnical problems such as those associated with false positive signalsfrom non-specific amplification and/or hybridization.

The present invention comprises a method for detecting a target nucleicacid sequence, comprising obtaining isolated nucleic acid sequences froma sample suspected of containing a target nucleic acid sequence;contacting a protection molecule with the nucleic acid sequences underhydridizing conditions sufficient to form a PNAS; and detecting thePNAS. The methods may further comprise the steps of digesting theisolated nucleic acids containing one or more PNAS with nucleolyticenzymes to form a PNAS/tail; and hybridizing a capture molecule to thePNAS/tail; prior to the step of detecting the PNAS. Additionally, themethods may further comprise the step of hybridizing of a reportermolecule to the PNAS/tail; prior to the step of detecting the PNAS. Amethod for detecting specific nucleic acid sequences, comprisingobtaining isolated nucleic acid sequences from a sample suspected ofcontaining a target nucleic acid sequence; contacting a protectionmolecule with the nucleic acid sequences under hydridizing conditionssufficient to form a PNAS; digesting the isolated nucleic acidscontaining one or more PNAS with nucleolytic enzymes to form aPNAS/tail; hybridizing a capture molecule to the PNAS/tail; hybridizingof a reporter molecule to the PNAS/tail; and detecting the PNAS.

The present invention comprises compositions for detecting specificnucleic acid sequences, comprising a protection molecule capable ofbinding with a specific nucleic acid sequence. A composition of thepresent invention may further comprise a capture molecule. Additionally,a composition of the present invention may further comprise a reportermolecule.

The methods and compositions of the present invention should be idealfor the detection of viruses and other microorganisms such as pathogensof humans, animals and plants, as well as genetic analysis ofpolymorphic gene sequences such as HLA typing. The methods of thepresent invention can be used in forensics, paternity determinations, ortransplantation or organs or tissues, or genetic disease analysis.

Accordingly, it is an object of the present invention to provide methodsto detect specific genetic sequences.

It is yet another object of the present invention to provide methods fordetecting specific DNA sequences involving triplex nucleotidestructures.

It is another object of the present invention to provide methods fordetecting specific RNA sequences involving triplex nucleotidestructures.

It is yet another object of the present invention to provide methods fordetecting specific DNA sequences involving duplex nucleotide structures.

It is another object of the present invention to provide methods fordetecting specific RNA sequences involving duplex nucleotide structures.

It is another object of the present invention to provide methods fordetecting specific RNA sequences involving PNA structures.

It is another object of the present invention to provide methods fordetecting specific DNA sequences involving PNA structures.

It is yet another object of the present invention to provide methods fordetecting specific DNA sequences involving antibodies.

It is yet another object of the present invention to provide methods fordetecting specific RNA sequences involving antibodies.

Another object of the present invention is to provide a method ofdetecting nucleic acid sequences involving radioactive labeled nucleicacids.

It is another object of the present invention to provide a method ofdetecting nucleic acid sequences involving non-radioactive labelednucleic acids.

It is yet another object of the present invention to provide a method ofdetection of specific genetic sequences with variable levels ofspecificity.

Another object of the present invention is to provide a method ofdetecting nucleic acid sequences for the determination of the identityof microorganisms.

It is another object of the present invention to provide a method ofdetecting nucleic acid sequences for the determination of the identityof human pathogens.

It is yet another object of the present invention to provide a method ofdetecting nucleic acid sequences for the determination of the identityof animal pathogens.

It is yet another object of the present invention to provide a method ofdetecting nucleic acid sequences for the determination of the identityof plant pathogens.

It is another object of the present invention to provide a method ofdetecting nucleic acid sequences for the determination of the geneticrelationship, such as paternity or species identification, of a sample.

It is yet another object of the present invention to provide a method ofdetecting nucleic acid sequences for the determination of potentialdonors of organs or tissues for transplantation purposes or forprotecting the blood supply.

It is another object of the present invention to provide a method ofdetecting nucleic acid sequences for use in forensic determinations.

It is yet another object of the present invention to provide a method ofdetecting nucleic acid sequences for the analysis of genetic diseases.

It is another object of the present invention to provide methods fortesting body or tissue fluids to detect microorganisms or otherpathogens.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWING

FIGURE 1 shows the steps of a method of the present invention. There arefive individual steps in the TPA procedure as shown in FIGURE 1: DNAisolation; PNAS formation, which in FIGURE 1 is a triplex formation;endo/exonuclease digestion; addition of the capture probe with anaffinity molecule, which in FIGURE 1 is didoxigenein; isolation of thePNAS/tails with capture probe by addition of magnetic beads; addition ofthe reporter probe with its label, which in this case, the label is FITC(fluorescein isothiocyanate) and detection of the labeled PNAS/tail withreporter and capture probes.

DETAILED DESCRIPTION

The present invention includes methods for the detection of a specifictarget nucleic acid sequence using a protection molecule that forms aprotected nucleic acid sequence (PNAS) structure including the targetnucleic acid sequence. An assay using the methods of the presentinvention may be referred to as TPA, target protection assay. Oneembodiment of the present invention is a method for the detection ofspecific DNA sequences. The present invention also includes methods forthe detection of specific RNA sequences. In the disclosure herein, thenucleic acid DNA will be used but it is to be understood that anynucleic acid, including RNA, can be used with the methods of the presentinvention. Where specific nucleases are referred to, any nuclease thatcan perform the specified function can be substituted for the namednuclease.

The steps of a method of the present invention, the Target ProtectionAssay (TPA), involves the combination of several techniques to arrive ata unique nucleic acid diagnostic tool that is specific for a targetnucleic acid sequence. A preferred method of the present invention,directed at a DNA target nucleic acid, involves the steps of 1) DNAisolation; 2) formation of the PNAS; 3) enzymatic digestion ofunprotected DNA; 4) capture; and 5) labeling and 6) detection of thePNAS.

Many of the individual procedures summarized herein may use techniquesknown to those skilled in the art of molecular biology, with severalvariations taught in the literature, or commercially available in theform of kits. It is to be understood that the present invention is notlimited by the specifically disclosed techniques, but any techniquesthat are capable of performing the same function or result can besubstituted for the ones described. For the purpose of example, a singletechnique will be described for each step in the TPA procedure. Suitablealternative techniques are noted where appropriate. However, the presentinvention is not to be limited thereby as many other suitablealternatives are intended to be included within the scope of theinvention.

The present invention includes within its scope such nucleic acidtargets as DNA (single and double stranded) and RNA (single and doublestranded). The methods of the present invention are useful forspecifically detecting the presence of very low copy number nucleic acidtargets in a vast excess of non-target nucleic acids.

The methods of the present invention involve protecting the targetnucleic acid sequence from nuclease attack with the protection molecule,a molecule such as a single stranded DNA or RNA or a peptide nucleicacid (PNA). The protection molecule is selected or designed to bindspecifically to the target nucleic acid sequence. The protectionmolecule, in association with the target nucleic acid sequence, forms astructure, the PNAS. For example, the PNAS includes, but is not limitedto, triplex and duplex nucleic acid structures, peptide nucleic acid andantibody associated structures.

The methods of the present invention may include a protection moleculeassociated with an affinity molecule that allows binding of the PNAS insolution to a fixed substrate. The presence of the affinity moleculepermits the removal of the excess extraneous nucleic acid. The methodsof the present invention further involve a reporter molecule to permitvisualization of the presence of the target nucleic acid.

An assay of the present invention that allows for the lowest level ofspecificity involves the binding of the protection molecule to thespecific target nucleic acid sequence to form the PNAS. Diagnostictechnologies are valuable only if they achieve high specificity (fewfalse positives) and high sensitivity (few false negatives). In order toprovide for a higher level of specificity, the present inventioncomprises methods wherein the target nucleic acid sequence is protectedfrom nuclease attack when bound by the protection molecule to form thePNAS, and one or two enzymatically generated 5' DNA tails are generated,on one or both sides of the PNAS for hybridization with a capture probecontaining an affinity molecule. The capture probe is selected ordesigned to specifically bind to a tail region of a tail-containingPNAS. This assay results in two levels of specificity-binding of theprotection molecule and binding of the affinity molecule to the tail ofthe target nucleic acid. The affinity molecule allows for the attachmentof the entire protection structure with the bound affinity molecule tobe attached to a fixed substrate. In this example of an assay with twolevels of specificity, either the affinity molecule or the protectionmolecule are labelled with any type of label known to those skilled inthe art. The label would allow for detection of the PNAS having anaffinity molecule.

A third level of specificity can be added to the assays contemplated bythe present invention by generation of two different tail regions,preferably one on each side of the target nucleic acid, that extendbeyond the target nucleic acid sequence bound by the protectionmolecule. The tail regions are included in the protected structure butare not bound to the protection molecule and the structure is named thePNAS/tails. One tail region could be bound to the capture probe toanchor the target to a fixed substrate (via the affinity molecule), andthe other tail used to bind a reporter probe with label to visualize thepresence of the nucleic acid target. The tail regions of the PNAS may ormay not be necessary for use depending on the level of specificitydesired. Preferrably, the capture probe and the reporter probe areselected or designed to bind specifically and exclusively to one tail orthe other, thereby ensuring that each of the two probes hybridizes tothe PNAS/tail.

Increasing the number of levels of specificity increases the specificityof the assay (no false positives), however, excessive levels ofspecificity may decrease the levels of sensitivity generated (high falsenegatives). The present invention comprises assays that are dynamicdiagnostic technologies that can be customized to deal with any specificnucleic acid target and yield any of a variety of desired levels ofspecificity.

Nucleic Acid Isolation

Nucleic acids may be isolated using any methods known to those skilledin the art. Nucleic acids, as used herein, means both DNA and RNA in allits forms found in cells or constructed by molecular biologicaltechniques.

The method for DNA isolation used will largely depend on the amount andtype of material to be extracted. Virtually any DNA isolation procedurereported in the literature which produces genomic or mitochondrial DNA,or any commercially available DNA isolation kit will suffice. The methodcontemplates that the sample amount of DNA to be used in each assay isconcentrated in a volume that can range from 0.1 to 1.0 mL depending onthe solubility of the DNA being tested. Larger DNA samples may requireuse of greater sized volumes. The methods of the present invention maytest amounts of sample nucleic acids between picogram amounts tomilligram amounts. The reactions components would have to be adjusted,for example, to provide adequate amounts for hybridization of thecomponents. The reaction components are proportionate to not only thesize of the sample tested but also to the relative number of targetsequences that are present. It is to be understood that the amount ofsample DNA will depend on the size and kind of sample.

The DNA is placed in a buffer suitable for the formation of PNAS, suchas a duplex or triplex structure using a duplex or triplex formingoligonucleotide (DFO or TFO) or a peptide nucleic acid. Many procedureshave been reported for the isolation of high molecular weight DNA fromseveral sources including whole blood, isolated blood cells, serum andplasma, fresh, frozen or prepared tissues, and tissue culture cells.

RNA can also be isolated by any methods known to those in the art.Published RNA isolation protocols lyse the cell in a chemicalenvironment that denatures ribonucleases, and fractionates the RNA typeof interest from other RNAs and other cellular macromolecules. The RNAisolation method used is dependent upon the cell type from which the RNAis isolated and the eventual use of the RNA.

There are published methods for preparing total RNA from eukaryaticcells, and such methods are herein incorporated by reference. InFavaloco, et al., 1979 and Chomczynski and Sacchi, 1987, cells are lysedusing guanidinium isothiocyanate. This method has few manipulations andyields clean RNA from many sources, and is the method of choice fortissues that have high levels of endogenous RNAse. In the third methodof Palmiter, 1974, cells are lysed with phenol and SDS. This results inclean, high molecular weight RNA from large quantities of plant cellsand also works well with some mammalian cells and tissues.

Published methods for preparing total RNA from prokaryotic cellsinclude: protocols for extracting RNA from gram-negative andgram-positive bacteria, using protease digestion and organic extractionto remove protein and nuclease digestion to remove DNA (Reddy, et al.,1990); and a simple protocol for rapidly isolating RNA from E.coliwithout organic extractions, protease, or nuclease treatment (Summers,1970). Lastly a published method (Aviv and Leder, 1972) can fractionatemessenger RNA from ribosomal and transfer RNA based upon the exclusivepresence of poly (A) tails on mRNA.

PNAS Formation

After isolation of the nucleic acid, the next step in the methods of thepresent invention include formation of the PNAS. This step introducesthe first level of specificity to the assay. This step involves theformation of the PNAS using a target nucleic acid sequence-specific TFOor DFO or PNA. Hereinafter, TFO will be used in the example of apreferred embodiment, but it is to be understood that triplex and duplexstructures and PNA are contemplated by the present invention.

The sequence of the TFO will depend on the specific target sequence tobe detected. The most well characterized triplex structure is the oneformed between a double stranded homopurine-homopyrimidine helix and asingle stranded homopyrimidine tract. Formation of such structures arewell known in the art. Specific details of the formation of suchstructures are given in the following references which are hereinincorporated by reference. S. W. Blume, J. E. Gee, K. Shrestha, and D.M. Miller. Triple helix formation by purine-rich oligonucleotidestargeted to the human dihydrofolate reductase promoter. Nucl. Acids Res.20: 1777-1784 (1992).

In this first type of triple helix, the third homopyrimidine strandbinds to the major groove, parallel to the homopurine strand of theWatson-Crick double helical DNA via Hoogstein hydrogen bonding. Thethird-strand thymidine (T) recognizes adenine-thymine (A:T) base pairsforming T:A:T triplets, and the third strand cytosine (C), protonated atthe N-3 position, recognizes guanidine-cytosine (G-C) base pairs formingC⁺ :G:C triplets. Homopyrimidine oligonucleotide have been shown to formlocal triplexes with corresponding homopurine sites in largerdouble-stranded DNAs. An alternative triplex structure is a doublestranded homopyrimidine-homopurine helix and a single strandedhomopurine tract (TFO). Yet other alternative triplex structurescomprise a combination of the two described structures.

The design of the TFO will generally follow thePyrimidine-Purine-Pyrimidine binding rules described previously, or maybe designed to form Purine--Purine-Pyrimidine triplexes if necessary.Such structures are well-known in the art. However, other binding motifsalso apply, examples: I. Rec A Mediated TFO binding in 4 base regions(Rec A required to remain in solution); II. Triple purine and triplepyrimidine triplexes. Rec A is a recombinant enzyme that catalyzes therecombination between two DNA strands with similar homology.

While not wishing to be bound by the following theory, the premise ofusing TFOs to select specific regions of DNA for diagnostic use requiresone to have a conserved sequence of DNA from the target sequence and tohave a long enough sequence to ensure hybridization and selectivity. Thehuman genome has approximately 5×10⁹ base pairs of DNA. In order to havea unique sequence this would require an oligonucleotide of approximatelybetween 16-20 nucleotides long. The actual number is probably smallerdue to the presence of intron sequences in the DNA. Longer sequencesincrease the hybridization between the TFO and DNA while decreasing thespecificity.

The selection of the TFOs is based on an empirical search forpoly-purine/pyrimidine stretches in the target region. In the methods ofthe present invention, several confounding factors such as DNA/proteininteractions should not interfere with the binding of the TFO to itstarget sequence. Also, secondary structure can be influenced bytemperature, which should allow for more efficient TFO binding. Oftenthe sequence is not entirely a homopurine strand but contains intermixedpyrimidines. Even though the introduction of pyrimidines could lower theTFO's affinity for the duplex DNA, the entire sequence still allowsselective binding at the proposed hybridization temperature. Theconditions to form the triplex structure may also vary depending on thetarget sequence, but must be compatible with the nucleases used in thesubsequent step. For example, the conditions may need to be adjusted foractivity by Exonuclease III (Exo III) and the restriction endonucleaseschosen for the next step in the procedure (see next section).

To aid in the formation of a triplex structure, a low pH buffer (pH6.8-7.4) would be optimal. This would also serve to help stabilize thestructure during the enzymatic digestion step. Additional stabilizationprocedures, known to those in the art, can also be employed. Forexample, while TFOs may work well under a variety of situations, thereare two fundamental problems unique to triplex formation. One is that,for the CT motif, acidic pH is required for triplex formation. Thesecond is that the recognition sequence is limited to oligopurines. Thefirst problem can be approached by altering the nucleic acid withchemical modifications, such as those taught in the art. See J. S. Lee,L. J. Woodsworth, P. Latimer, and A. R. Morgan.Poly(pyrimidine).poly(purine) synthetic DNA's containing5-methylcytosine form stable triplexes at neutral pH. Nucleic Acids Res.12: 6603-6614 (1984). This is done by replacing dC with modified basessuch as 5-methyl-dC, C-5 propyne pyrimidine,6-methyl-8-oxo-2'-deoxyadenosine, or 2'-O-methylpseudocystein.

Another approach is to add a linker to increase and stabilize theinteraction with the target sequence. In an additional approach the TFOcan also be conjugated to unique chemical groups to allow the formationof a triplex structure when it normally would not. Not only cantriple-stranded DNA complexes be stabilized by a high ionic strength orby the presence of cations like magnesium, but also by triple-helixspecific ligands called benzopyridoindole (BPI) derivatives, whichintercalate in triple helix complexes. The present inventioncontemplates all of these methods that are well known in the art andother binding schemes that function in the same manner.

Lower pH conditions are compatible, although not necessarily optimal,with Exo III and most restriction endonucleases. In addition, theseconditions allow triplex formation at the elevated temperatures (37° C.)needed for the subsequent digestion step.

An example of formation of a triplex structure is given here. A >10-foldmolar excess of the TFO is added to the isolated DNA (10 pmoles TFO/μgDNA) and the triplex structure is allowed to form for 10 min. When theDNA and TFO are mixed in equal amounts, the kinetics of triplexformation has been characterized by half-decay times (t1/2) of 150-390seconds. By contrast, when the TFO was in ten-fold excess over the DNAthe kinetics were faster and the t1/2 decreased to 19-28 seconds. Therate of triplex appears to be about three orders of magnitude slowerthan the rate of duplex recombination, which has a rate constant in theorder of 10⁶. The apparent activation energy associated with the rateconstant of triplex formation was small and negative (E₁ =26±15 kJ/mol).The first order rate constant of triplex formation (k₋₁) depends ontemperature and was in the range of 10⁻⁷ to 10⁻⁵ s⁻¹ (at 20° C. and 33°C., respectively), with an apparent activation energy that was large andpositive (E₋₁ =355±33 kJ/mol). The rate of triplex formation also showeda dependence on ionic strength (I) of the buffer solution (17,23,24). Adecrease of I from 137 mM to 57 mM resulted in a six-fold decrease inthe association constant.

Enzymatic Digestion of DNA

This step in the methods of the present invention assures that 5' tailsof approximately at least 20 base pairs are generated upstream anddownstream from the PNAS. These tails are useful for the capture anddetection steps. This step also ensures that all non-specific nucleicacids are digested as well as unbound TFO, DFO and PNA molecules, thusreducing potential false-positive signals.

More specifically, once the PNAS is formed and stabilized, a mixture ofexo- and endonucleases are added to the mixture. The endonucleases aresequence specific restriction enzymes chosen to flank the target nucleicacid site, leaving approximately 20 base pairs (usually more) of nucleicon each side. Where the target nucleic acid sequence is dsDNA, theexonuclease must be ds DNA dependent which digests only one strand(either 3' to 5' or 5' to 3'), leaving large tracts of ss DNA availablefor hybridization with specific probes. A preferred enzyme (and the oneused in all examples) is Exo III. Exo III is a monomeric protein of28,000 Daltons that catalyzes the stepwise 3' to 5' removal of5'-mononucleotides from ds DNA with a free 3'-OH end. Exo III alsocontains an inherent 3' phosphatase activity and a RNAse H activity.Thus, Exo III can also be used in methods of the present invention thatuse RNA target sequences.

The enzymes shown in Table 1 may be used in the present invention. Thepresent invention is not limited to the disclosed enzymes.

                                      TABLE 1                                     __________________________________________________________________________    Properties of some mammalian nucleases                                                       Mode of     Reaction                                           Enzyme Substrate                                                                             action.sup.1                                                                        pH.sup.2                                                                         Mg.sup.1+                                                                        product.sup.3                                                                       Mol. wt.                                     __________________________________________________________________________    DNAse I                                                                              ds/ss DNA                                                                             Endo  7.1                                                                              +  5' oligos                                                                           31 Kdal                                      DNAse II                                                                             ds/ss DNA                                                                             Endo  4.1                                                                              -  3' oligos                                                                           38 Kdal                                      DNAse III                                                                            ss Duplex DNA                                                                         Exo   8.5                                                                              +  5' monodinu-                                                                        52 Kdal                                                                 nucleotides                                        DNASe IV                                                                             Duplex DNA                                                                            Exo 3'5'                                                                            8.5                                                                              +  5' mono                                                                             42 Kdal                                      DNAse V                                                                              Duplex DNA                                                                            Exo 3'5'/5'3'                                                                       8.8                                                                              +  5' mono                                                                             12 Kdal                                      DNAse VI                                                                             Ss DNA  Endo  9.5                                                                              +  5' oligo                                                                            45 Kdal                                      DNAse VII                                                                            ss and nicked &                                                                       Exo 3'5'                                                                            7.8                                                                              +  5' mono                                                                             43 Kdal                                             ds DNA                                                                 DNA VIII                                                                             5' ss and nicked                                                                      Exo 5'3'                                                                            9.5                                                                              +  5' oligos                                                                           31 Kdal                                      Correxo                                                                              ss DNA nicked                                                                         Exo 3'5'/5'3'                                                                       8.0                                                                              +  5' oligos                                                                           30-35 Kdal                                          UV'd ds DNA.sup.4                                                      Lysosomal or                                                                         RNA or DNA                                                                            Exo 5'3'                                                                            5.5                                                                              -  3' mono                                                                             70 Kdal                                      spleen with 5' OH                                                             exonuclease                                                                   __________________________________________________________________________     .sup.1 Endo = endonucleolytic. Exo = exonucleolytic                           .sup.2 optimum pH                                                             .sup.3 Oligonucleotide shown is the main reaction product.                    oligos = oligonucleotides                                                     mono = mononucleotides                                                        .sup.4 UV irradiated double stranded DNA                                 

Exo III is commercially available from many sources at a reasonablecost, and will create the desired single stranded regions adjacent tothe target DNA. Most importantly, Exo III will not digest dsDNA that isin a triplex structure, and thus the PNAS with the target sequence willbe protected from digestion. One unit of Exo III will digest 50 ng ofgenomic DNA at 37° C. in 10 min. The main purposes of the endonucleasesis to produce free ds DNA ends close to the TFO target site to aid theEXO III in digesting the genomic DNA and tail generation. Furthermore,endonuclease activity will increase the solubility of the sample DNA andcomplete digestion would eliminate nontarget DNA as a source ofnon-specific interactions. In some reactions, pretreatment withnoninterfering nucleases may be used to increase the nucleic acidsolubility and help minimize the solution volume to be tested. Thisshould allow the use of less Exo III than would be required to digestfull length genomic DNA. In addition, complete endonuclease digestion isalso not necessarily required to obtain the desired product.

In its simplest form, methods of the present invention can be fulfilledby this single protection step by concomitantly introducing a moleculefor the capture system and a reporter molecule for target identificationof the PNAS, yielding an assay with a single level of Respecificity.Additional nuclease steps may be necessary to prevent interference fromunbound TFO and non-specific signals. In order to increase the level ofthe specificity, additional steps involving additional oligonucleotideprobes can be added.

Oligonucleotide Probe--Capture System

Further steps in the methods of the present invention comprise thesecond level of specificity. These steps involve the hybridization of acapture probe containing an affinity molecule (such as biotin ordigoxigenin) to the digested PNAS/tail and binding of the complex to aderivatized solid support (such as magnetic beads, microtiter plates, ormembranes). This step allows greater sample manipulation because it canbe used for concentration of the target sequences, buffer exchange, aswell as removal of non-target nucleic acids.

The sequence of the capture probe will be complementary (Watson-Crickbase pairing) to one of the ss (single-stranded) DNA regions flankingthe PNAS which was generated by the nuclease digestion step. Forexample, a greater than 10-fold molar excess of the capture probe can beadded to the PNAS/tail under conditions favoring specific hybridization.Such conditions are known to those skilled in the art. For example, 2.0MNaCl, 0.2 M sodium acetate, pH 4.5, 50° C., for 1 hour could be used.Following hybridization, the complexes will be purified by co-incubationwith the derivatized solid support for an additional 1 hour under thesame conditions, followed by adequate washing of unbound complexes (e.g.8 times with hybridization buffer).

At this point, the complexes may be dissociated from the support, ifdesired, with a dissociation buffer. Such conditions are known to thoseskilled in the art. For example,1.0 M Tris-HCl, pH 9, 0.5 mM EDTA for 20min. could be used.

The options for affinity capture systems are numerous and are well knownin the art. Such capture systems include, but are not limited to, thetwo most cited systems, biotin (capture with streptavidin) anddigoxigenin (Dig, Boehringer-Mannheim, captured with anti-Dig antibody).However, any other similar system can be used.

In the case of solid supports, the situation is similar. The use ofderivatized membranes (such as nylon) have had widespread application inthe literature, and could be used in the present invention wheredetection using film exposure or phosphor imaging (such as withradioactivity or chemiluminescence) is desired. These supports also workwell with the available enzyme conjugate systems (alkaline phosphatase[AP] or horseradish peroxidase [HRP]) with non-radioactive colorproducing substrates.

Another option for a solid support is a derivatized microtiter plate.These plates are available with many options from several sources. Oneadvantage of microtiter plates is the availability of many supportingsystems for automated manipulation (i.e. washing steps) and detectionoptions (radioactivity, U.V. and visible light spectroscopy, andfluoresence). This system has the disadvantage of being limited to arelatively small volume (100-200 μl/well).

A system that is rapidly growing in popularity is the use of derivatizedmagnetic beads (Dynal). Non-magnetic beads (usually agarose orsepharose) have been used for affinity capture and purification for manyyears. The magnetic bead system is a preferred system for themanipulations needed for the methods of the present invention, and itwill be the system used for the example here. These beads are availablederivatized with both strepavidin and anti-Dig.

The assay could be completed at this point if this level of specificityis acceptable. The capture probe or protection molecule could be labeledso that the captured PNAS could be detected.

Oligonucleotide Probe Detection

The third level of specificity in the methods of the present inventionis achieved through the use of a reporter probe. It is also at this stepthat the specific mechanism of detection is introduced. The reporterprobe consists of a synthetic single stranded oligonucleotidecomplementary to the opposite single stranded end (not being used forattachment to the capture system) generated by the nuclease digestion.The composition of this detection step will vary depending on the methodused. All methods of detection will require the presence of a reporterprobe that be specifically detected as it binds to a specific sequenceon the captured PNAS. For this invention the method need only besufficiently sensitive to detect this specific probe-complex interactionso that a positive results can be defined.

The composition of the oligonucleotide probe will depend on the methodof detection used. For direct detection of the probe, the probe maysimply be a specific sequence of nucleotides complimentary to thespecific sequence on the PNAS/tail where the interaction is detected byany physical method that can detect a specific interaction ofoligonucleotides. An example of such a detection technique would befluorescence anisotropy where the relative amount of bound probe can bemeasured directly without the removal of unbound probe.

In the case of fluoresence anisotropy, the relative level of bound probecan be measured directly without the removal of unbound probe. Methodsbased on separation might perturb the equilibrium binding of the probeand may led to erroneous results. In the use of anisotropyspectrophotometric determination, the concentration of free and boundmaterial are measured by an observable change in the chromophore (i.e.due to changes in the molecular weight after hybridization). Thefraction bound can be expressed as f_(b) =(r_(obs) -r_(in))/(r_(b)-r_(in)), where f_(b) is the fraction bound, r_(in) is the initialanisotropy, r_(obs) is the observed anisotropy after hybridization, andr_(b) is the total binding (determined by titrating a smallconcentration of the probe with an excess of binding agent). With thisinformation, the kinetics of binding can be seen for both smallmolecules and macromolecules. This methodology has been applied toobserving oligonucleotide hybridization in solution, and is used in theTPA assay.

Other physical methods may include evenescent wave technology thatdetects changes in the physical properties of a surface as proteins ornucleic acids specifically interact on that surface. There are a numberof related physical methods that can be used where the specificinteraction can be measured without separation of bound and free labeledoligonucleotide.

Direct detection of the oligonucleotide probe can involve a specificsequence of nucleotides complimentary to the 5'tail on the PNAS/tailmoiety where the oligonucleotide is derivatized with a label that canemit a signal when specifically bound to the target DNA Triplex. For thedetection to be specific, any unbound directly labeled oligonucleotidewould have to be separated from the bound form prior to detection.Examples of labels that can be directly incorporated intooligonucleotides include: radioactive isotopes, such as ³ H, ¹⁴ C, ³²P,¹²⁵ I that are detected using scintillation or gamma counters,fluorescent dyes that can be detected by fluorimeters, bioluminescent,chemiluminescent or electrochemiluminescent labels that can be detectedusing specific triggering reactions to generate light that can bequantified in a luminometer.

The various types of labels and methods of labeling nucleotide sequencesare well known to those skilled in the art. Many of these labelingformats can be used in the above described assays with the first orsecond level of specificity. Several specific labels or reporter groupsare set forth below.

For example, the label can be a radiolabel such as, but not restrictedto, ³² P, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, or ¹³¹ I. A ³² P label can beincorporated into the sequence of the probe by nick-translation,end-labeling or incorporation of labelled nucleotide. A ³ H, ¹⁴ C or 35Slabel can be incorporated into the sequence of the probe byincorporation of a labelled precursor or by chemical modification. An¹²⁵ I or ¹³¹ I label can be incorporated into the sequence of the probeby chemical modification. Detection of a label can be by methods such asscintillation counting, gamma ray spectrometry or autoradiography.

The label can also be a Mass or Nuclear Magnetic Resonance (NMR) labelsuch as, for example, ¹³ C, ¹⁵ N, or ¹⁹ O. Detection of such a label canbe by Mass Spectrometry or NMR.

Dyes and fluorogens can also be used to label the probes. Examples ofdyes include ethidium bromide, acridines, propidium and otherintercalating dyes, and 4',6'-diamidino-2-phenylindole (DAPI)(SigmaChemical Company, St. Louis, Mo.) or other proprietary nucleic acidstains. Examples of fluorogens include fluorescein and derivatives,phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, Texas Red orother proprietary fluorogens. The fluorogens are generally attached bychemical modification. The dye labels can be detected by aspectrophotometer and the fluorogens can be detected by a fluorescencedetector.

The probe can alternatively be labelled with a chromogen to provide anenzyme or affinity label. For example, the probe can be biotinylated sothat it can be utilized in a biotin-avidin reaction which may also becoupled to a label such as an enzyme or fluorogen. The probe can belabelled with peroxidase, alkaline phosphatase or other enzymes giving achromogenic or fluorogenic reaction upon addition of substrate. Forexample, additives such as 5-amino-2,3-dihydro-1,4-phthalazinedione(also known as Luminol™) (Sigma Chemical Company, St. Louis, Mo.) andrate enhancers such as p-hydroxybiphenyl (also known as p-phenylphenol)(Sigma Chemical Company, St. Louis, Mo.) can be used to amplify enzymessuch as horseradish peroxidase through a luminescent reaction; andluminogeneic or fluorogenic dioxetane derivatives of enzyme substratescan also be used.

Recognition sites for enzymes, such as restriction enzyme sites, canalso be incorporated into the probes to provide a detectable label. Alabel can also be made by incorporating any modified base or precursorcontaining any label, incorporation of a modified base containing achemical group recognizable by specific antibodies, or by detecting anybound antibody complex by various means including immunofluorescence orimmuno-enzymatic reactions. Such labels can be detected usingenzyme-linked immunoassays (ELISA) or by detecting a color change withthe aid of a spectrophotometer. It will be understood by those skilledin the art that other reporter groups can also be used.

Indirect detection of the oligonucleotide probe can involve a specificsequence of nucleotides complimentary to the specific sequence on thePNAS/tail where the oligonucleotide is derivatized with a reagent orentity that can be caused to produce a detectable signal in the presenceof another specific reagent or entity. An example of an indirectdetection system is the covalent derivatization of the oligonucleotideprobe with a unique chemical structure that can be uniquely recognizedby a binding partner; i.e., a hapten label such as biotin or digoxigeninor a unique piece of nucleic acid or nucleic acid related material whereavidin, anti-digoxigenin, or a complimentary strand of nucleic aciditself is directly labeled and capable of detection by a physical methodafter removing any free label from specifically bound label. Anotherexample of an indirect label is an oligonucleotide that is covalentlyderivatized with an enzyme that can convert a substrate into adetectable compound or release energy that can be detected by physicalmethods. Examples of enzyme-substrate pairs that can be used forindirect detection include:

1) Phosphatases such as alkaline phosphatase that can be detected byaddition of phosphorylated compounds which when dephosphorylated by theresult in compounds that, a) absorb light at a wavelength different formthe substrate; b) can produce a specific fluorescence; c) becomeluminescent; d) become a substrate for a second enzyme that can beincluded with a second substrate to generate a detectable signal.

2) Peroxidases, for example, horseradish peroxidase, whose reactionproducts in the presence of appropriate compounds can generate compoundsthat, a) absorb light at a wavelength different form the substrate; b)can produce a specific fluorescence; c) become luminescent; d) become asubstrate for a second enzyme that can be included with a secondsubstrate to generate a detectable signal.

3) Luciferases that can be detected by addition of appropriatesubstrates and cofactors which result in the production of light.Alternatively, luciferases can be included as the second enzyme inassays where the substrate was a phosphorylated luciferin that is onlyacted upon by a luciferase after removal of the phosphate. Otherhydrolytic enzymes other than the specific ones listed here can be usedas indirect enzyme labels.

The methods of the present invention can be used to detect single copyor low copy number nucleic acid sequences from any size sample,including large amounts of nucleic acids. An unexpected benefit of theassays of the present invention resides in the ability to process largesamples of nucleic acid and to detect and quantify specific nucleotidesequences that make up only a minor component of the complex mixtures ofsequences in the large sample.

The sensitivity limits can be approximated by evaluation of availabledetection systems combined with the amount of target that can beobtained from a specific sample size. A very sensitive system fornucleic acid detection is a bioluminescence technique based on thephotoprotein, AquaLite®. This technique is described in Actor etal.,(1996) J. NIH Res. 8 (10):62, herein incorporated by reference in itentirety. The system is capable of detecting 3×10⁶ specific sequences ofDNA in a hybridization immunoassay technique with high signal tobackground noise ratio.

In the methods of the present invention, a bioluminescent conjugate ofAquaLite®, coupled to an anti-digoxigenin antibody, is used to detect adigoxigenenin labeled reporter probe containing 2-3 digoxigeninmolecules used in the methods of the present invention. At the presenttime, the lower limit of detection of the signal produced by thebioluminescence protein requires that there be 3×10⁶ signals produced.Amplification systems, such as PCR would require amplifying a selectedsequence to reach this level of detection. In contrast, using themethods of the present invention one could start with a large originalsample that contains at least 3×10⁶ specific sequences and detect themdirectly from the large sample.

The limiting step is the signal detection system, not the assay of themethods of the present invention. Other techniques may be used toprovide lower limits of detection. With a signal amplification system incombination with TPA, single copy genes could be detected DNA samplesfrom as little as 100 μl of a blood sample.

For example, a very early detection of infection with HIV could be madewith the methods of the present invention. Without TPA, the earliestdetection of HIV could not occur until the infected person producedantibodies to HIV, a period of 6 months after initial infection. UsingTPA, the blood could be tested immediately after possible HIV infectionby isolating all white blood cells via leucophoresis, then extractingthe DNA, (approximately 5-8 mg DNA/500 mL of whole blood), assaying withmethods of the present invention using a labeled reporter probe with 2-3digoxigenin, and detecting the HIV sequences with AquaLite® coupled toan anti-digoxigenin antibody. Should there not be enough sequences forthe signal detection system in the initial sample, subsequent bloodsamples could be taken and pooled because TPA can be employed with sucha large sample size of nucleic acid. This testing procedure couldprovide very early detection of infection with HIV.

The present invention comprises a method for detecting a target nucleicacid sequence, comprising obtaining isolated nucleic acid sequences froma sample suspected of containing a target nucleic acid sequence;contacting a protection molecule with the nucleic acid sequences underhydridizing conditions sufficient to form a PNAS; and detecting thePNAS. The methods may further comprise the steps of digesting theisolated nucleic acids containing one or more PNAS with nucleolyticenzymes to form a PNAS/tail; and hybridizing a capture molecule to thePNAS/tail; prior to the step of detecting the PNAS. Additionally, themethods may further comprise the step of hybridizing of a reportermolecule to the PNAS/tail; prior to the step of detecting the PNAS. Amethod for detecting specific nucleic acid sequences, comprisingobtaining isolated nucleic acid sequences from a sample suspected ofcontaining a target nucleic acid sequence; contacting a protectionmolecule with the nucleic acid sequences under hydridizing conditionssufficient to form a PNAS; digesting the isolated nucleic acidscontaining one or more PNAS with nucleolytic enzymes to form aPNAS/tail; hybridizing a capture molecule to the PNAS/tail; hybridizingof a reporter molecule to the PNAS/tail; and detecting the PNAS.

The present invention comprises compositions for detecting specificnucleic acid sequences, comprising a protection molecule capable ofbinding with a specific nucleic acid sequence. A composition of thepresent invention may further comprise a capture molecule. Additionally,a composition of the present invention may further comprise a reportermolecule.

Procedure Variations in the Methods of the Present Invention

As discussed above, the methods of the present invention include a widevariety of alternative methods which can be substituted within each ofthe above described steps. An entire method could be performed in situ(using intact cells) and evaluated microscopically or in a flowcytometer. In addition, the steps themselves may also be modified toachieve the desired result. For example, Steps 2 (formation of the PNAS)and 3 (digestion of the extraneous nucleic acids) may be combined into asingle procedure. This could be accomplished because the conditions thatare described for the formation of the PNAS (Step 2) allow for rapidbinding of the protection probe to its target sequence (see above fortheory of triplex formation kinetics). As long as the formation of theprotection structure is significantly faster than the digestion of thenucleic acid by the exonuclease (Step 3), there will still be completeprotection of the protection structure with the target sequence. A leadtime of at least approximately 10 minutes for the formation in Step 2was included to insure that the advantage went to the binding of theprotection probe over that of enzymatic DNA digestion, however this maynot be required in most cases.

In this respect, Steps 4 and 5 could also be combined into a singlehybridization/capture step with no purification in between. Since eachprobe is unique to its own target sequence, there should be no danger ofcross hybridization to produce false signals. This possibility isfurther reduced by the fact that each probe carries a different label(i.e. capture with Dig vs. reporter with FITC). Since the hybridizationand wash procedures are identical in each step, combining the two wouldrepresent a significant simplification of the steps of the methods ofthe present invention. Ultimately the number of method steps isdependent on the desired level of specificity. Excessive steps may havea negative effect on sensitivity. Those skilled in the art would be wellaware of the level of specificity desired and the level at which theassay should be performed.

The methods of the present invention are especially useful for detectingspecific genetic sequences. The present invention comprises methods suchas the Target Protection Assay (TPA), which has the advantage ofallowing the processing of very large amounts (>1 mg) of purified DNA,thus eliminating the need for artificial amplification procedures suchas PCR, while enabling the detection of single target sequences. Inaddition, the three levels of specificity--target protection, captureprobe, and reporter probe--drastically reduce technical problems such asthose associated with false positive DNA amplification and/orhybridization signals.

The methods of the present invention can be used for the detection ofviruses and other microorganisms such as pathogens of humans andanimals, as well as genetic analysis of polymorphic gene sequences suchas with HLA typing. The methods of the present invention can be used fortaxonomical purposes for cells, microorganisms, animals, plants or anyother nucleic acid containing organisms. The isolation of specificnucleic acid sequences could be used for diagnosis of diseases found inhumans, animals, plants or other organisms. The methods of the presentinvention can be used in forensics, paternity determinations, ortransplantation or organs or tissues, or genetic disease analysis.Microbial nucleic acid sequences are defined at the nucleic acidsequences from microorganisms such as, but not limited to, viruses,bacteria, micoplasma, fungi, viroids, slow viruses, and scrapie-likeorganisms.

The methods of the present invention can be used for detection ofnucleic acid sequences and thus are applicable to many uses. Thefollowing is a list of uses of the methods of the present invention:

Testing the blood supply to prevent the transmission of infectiousagents

Detection of infectious agents in blood, blood products, and theorgan-donor supply

Detection of HIV status early in the course of infection

Confirmation of diagnosis of pediatric AIDS

Diagnosis of hereditary disease

Early detection of infectious diseases from fluids or tissues ofinfected humans, animals and plants.

Early detection of tumor cells in normal tissues

Detection of type I diabetes during fetal development

Determination of drug resistance prior to administration of the drug

Forensic identity testing

For example, the methods of the present invention can be used for thedetection of nucleic acids in samples taken from bodily fluids and fromenvironmental sources such as surfaces, air, or water. Because themethods of the present invention can isolate specific nucleic acidsequences from samples containing large amounts of nucleic acids, thesource of the nucleic acid is not to be limited by the examples hereintaught. Any source of nucleic acid can be employed with the methods ofthe present invention.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1 General Format of Target Protection Assay Using A ds DNATarget Sequence With PNAS Mediated By Triplex Formation

Isolation of DNA

The following protocol is a representative procedure for the rapidisolation of DNA from large amounts of whole blood: 150 mL of bloodcollected in venipuncture tubes (heparin, ACD or EDTA) is pooledtogether and diluted with 150 ml Isoton II (Coulter Diagnostics) in a500 ml centrifuge bottle. 30 ml of 10% Triton X-100 is added and mixedvigorously for 3 seconds. Cell nuclei are pelleted at maximum speed(12,000×g) for 5 minutes. After removal of the supernatant, the pelletis resuspended in 10 ml PK mixture (10 mM Tris-HCl, pH 8.0, 1 mM EDTA,0.5% Tween 20, 0.5% NP-40, and 2.5 mg/ml Protease K), incubated at 55°C. for 15 min, 95° C. for 10 min (to inactivate the Protease K), andthen slowly cooled to room temperature.. The sample is then transferredto a centrifuge tube and spun at 12,000×g for 10 minutes. Thesupernatant is recovered and the DNA is pelleted with the addition of0.2 volumes of 10M ammonium acetate and 2 volumes of ethanol. Theprecipitated DNA is pelleted at 5,000×g for 10 minutes, washed twicewith 70% ethanol, and then resuspended in 0.5 ml sterile water. Mildsonication or shearing may be required to obtain complete dissolution ofthe pellet. Approximately 1 mg of total genomic DNA should be recoveredfrom 150 ml whole blood (approx. 150 million nucleated cells). Any RNApreparative technique can also be applied.

Formation of PNAS mediated by triplex formation

To the 0.5 ml DNA sample in water, add 50 μl 10× TFO buffer (0.25 MTris-acetate, pH 7.0, 0.5 M NaCl, 100 mM MgCl₂, 50 mM -mercaptoethanol,0.10 mg/ml BSA, and 40 mM spermine-HCl), followed by 10 nmoles of thespecific TFO. Incubate for a period of time, and at a temperaturesufficient to permit the formation of stable PNAS, for example at 37° C.for 10 min, before proceeding to the next step.

Enzymatic Digestion

Add 500 units of each restriction enzyme (50 μl in most cases) and 4,000units of Exo III (40 μl of a 100,000 u/ml stock). Incubate the reactionat 37° C. for an additional 50 min, followed by inactivation of theenzymes by a biochemical or biophysical method. The sample is now readyfor the next step in the procedure.

Capture System

To the digested DNA mixture, add 10 nmoles of Dig labeled capture probeand 0.5 ml 2.5× hybridization buffer (5.0 M NaCl, 0.5 M NaOAc, pH 4.5).Incubate the mixture at optimal hybridization temperature for a periodof time sufficient to permit stable hydridization complexes to form, forexample 1 hour, followed by the addition of 100 μl of anti-Dig coatedmagnetic beads, washed and resuspended in hybridization buffer. After anadditional 1 hour incubation, isolate the beads using a magneticparticle concentrator and wash eight times with 0.5 ml hybridizationbuffer. The sample is now ready for the final step in the DNA triplexTPA procedure.

A FITC labeled reporter probe is used and detection is accomplishedusing fluorescence anisotropy. After the initial anisotropy of a 1.0 mLsolution containing 10 nmoles of reporter prove in hybridization bufferis measured, it is added to the washed magnetic beads. The mixture isincubated for 1 hour at 50° C. with gentle rocking, followed by transferof the entire contents (including beads) to an Abbott TDM sample vial.The anisotropy is then remeasured compared to the initial value foranalysis. The fraction bound can be expressed as f_(b) =(r_(obs)-r_(in))/(r_(b) -r_(in)), where f_(b) is the fraction bound, r_(in) isthe initial anisotropy, r_(obs) is the observed anisotropy afterhybridization, and r_(b) is the total binding (determined by titrating asmall concentration of the probe with an excess of binding agent).

EXAMPLE 2 HIV

Human immunodeficiency virus type 1 (HIV-1) is one of the two etiologicagents of AIDS. Currently, serologic assays which detect the presence ofanti-HIV antibodies are used to screen blood and blood products. Whilegenerally reliable, these tests will occasionally produce false positiveresults due to cross reactive antibodies or false negative results ifthe infection is at an early stage before the onset of a measurableimmune response. It is in the latter case that alternative methods suchas TPA (Target Protection Assay, a method of the present invention) maybe particularly useful, since large amounts of sample DNA may beprocessed and tested in a single assay tube. A direct assay for thevirus using co-cultivation with a susceptible cell line does exist,however this method is labor intensive and requires several days tocomplete. The following example will describe the extraction of a largeamount of blood for the worst case: that of a recently infectedindividual with low levels of infected CD4 positive cells.

1. Extract DNA from 150 ml whole blood (150 million white cells) asdescribed above in Example 1. Resuspend purified DNA in 0.5 ml water.

2. Add 50 μl 10× TFO buffer and 10 nmoles TFO:

HIV-1 TFO: (SEQ ID NO:1)

5'-TTT TCT TTT CCC CCC T-3'

3. Incubate 10 min at 37° C.

4. Add 500 units Sau 3A and 4,000 units Exo III.

5. Incubate 50 min at 37° C., followed by 20 min at 60° C.

6. Add 0.5 ml 2.5× hybridization buffer and 10 nmoles of Dig labeledcapture probe:

HIV-1 Capture probe: (SEQ ID NO:2)

5'-ACT GCC ATT TGT ACT GCT GT-Dig-3'

7. Incubate 50° C. for 1 hour.

8. Add 100 μl washed Dig coated magnetic beads.

9. Incubate 50° C. for 1 hour with rocking.

10. Place tube in a magnetic concentrator and remove liquid.

11. Wash 8× with 0.5 ml hybridization buffer.

12. Resuspend beads in 1.0 ml hybridization buffer containing 10 nmolesreporter probe previously measured for fluorescence anisotropy:

HIV-1 reporter probe: (SEQ ID NO:3)

5'-GAA TAG TAG ACA TAA TAG TA-FITC-3'

13. Incubate 50° C. for 1 hour.

14. Remeasure anisotropy and analyze fraction of bound probe (f_(b)) bythe formula given above.

Alternatively, after step 13 the beads can be repurified with themagnetic particle concentrator, washed 8× with hybridization buffer, andplaced in a fluorometer for direct fluoresence measurement (-_(exc) =490nm, -_(em) =520 nm), or the beads can be placed on a slide for viewingon a fluorescent microscope.

EXAMPLE 3 Borrelia bergdorferi

The spirochete B. bergdorferi is the causative agent of Lyme disease.This agent is transmitted primarily through the bite of infected ticks,resulting in arthritic, neurological, and rheumatoid symptoms, makingclinical diagnosis difficult. The primary tests for this agent areserologic and bacterial culture, both of which are relatively low insensitivity, especially at the early stages of the disease. Sources oftest material include whole blood, serum, joint fluid, cerebrospinalfluid, and urine. The following procedure is for 30 ml of whole blood:

1. Extract DNA from 30 ml whole blood (30 million white cells) asdescribed above in Example 1. Resuspend purified DNA in 0.5 ml water.

2. Add 50 μl 10× TFO buffer and 2 nmoles TFO: (SEQ ID NO:4)

TFO: 5'-TCC GCC TTT TOT TGT TTT TC-3'

3. Incubate 10 min at 37° C.

4. Add 100 units Ssp I, 100 units of Xho I, and 800 units Exo III.

5. Incubate 50 min at 37° C., followed be 20 min at 60° C.

6. Add 0.5 ml 2.5× hybridization buffer and 2 nmoles of Dig labeledcapture probe:

Capture probe: (SEQ ID NO:5)

5'-CCA GGC AAA TCT ACT GAA ACG CTG-Dig-3'

7. Incubate 50° C. for 1 hour.

8. Add 20 μl washed Dig coated magnetic beads.

9. Incubate 50° C. for 1 hour with rocking.

10. Place tube in a magnetic concentrator and remove liquid.

11. Wash 8× with 0.5 ml hybridization buffer.

12. Resuspend beads in 1.0 ml hybridization buffer containing 2 nmolesreporter probe previously measured for fluorescence anisotropy:

reporter probe: (SEQ ID NO:6)

5'-TAG ACA AGC TTG AGC TTA AAG-FITC-3'

13. Incubate 50° C. for 1 hour.

14. Remeasure anisotropy and analyze fraction of bound probe (f_(b)) bythe formula given above.

Alternatively, after step 13 the beads can be repurified with themagnetic particle concentrator, washed 8× with hybridization buffer, andplaced in a fluorometer for direct fluorescence measurement (-_(exc)=490 nm, -_(em) =520 nm), or the beads can be placed on a slide forviewing on a fluorescent microscope.

EXAMPLE 4 B. dermatitidis

B. dermatitidis represents a family of fungal pathogens who's incidenceof infection is increasing, especially among immunocompromised patients(such as organ transplant recipients). Current tests for fungalpathogens include serology and cultures, which are relatively slow andinsensitive. A DNA-based test (non-PCR) has also been reported, butrequires initial culturing of the pathogen before testing. The TPAprocedure for this pathogen is identical to the previous examples withthe following modifications: Isolate the DNA from 0.3 g wet yeasts ormycelia forms by the method of Lee and Taylor (39), and resuspend theisolated DNA in 0.5 ml water. Use 1 nmole of a TFO of the sequence (SEQID NO:7) 5'-TTC CTC CGT CGT CCG CGC-3' in the triplex formation step.Use 100 units of Rsa I and Msp I, and 800 units of Exo III in thedigestion step. Use 1 nmol of Dig labeled capture probe of the sequence(SEQ ID NO:8) 5-GGT AGC CGT TTC TCA GGC TCC TC-Dig-3', and 50 μl Digcoated magnetic beads for capture. Finally, use 1 nmol of a reporterprobe of the sequence (SEQ ID NO:9) 5'-GAG GTA GTG ACA ATA AAT ACTGAT-FITC-3' for the detection step.

EXAMPLE 5 Babesia microti

B. microti is a tick transmitted protozoal pathogen which infects humansand is found primarily in the U.S. This is the primary etiologic agentassociated with the Nantucket fever outbreak off the coast of NewEngland. Diagnosis is based mainly on serologic detection of anti-B.microti antibodies or the visualization of intraerythrocytic inclusions.The TPA procedure for this agent is identical to the above examples withthe following modifications:

1. Extract DNA from 30 ml whole blood and resuspend in 0.5 ml water.

2. Use 2 nmoles of each probe at the appropriate step:

TFO: (SEQ ID NO:10)

5'-GGG GCG ACG ACG GGT GAC GGG G-3'

Capture: (SEQ ID NO:11)

5'-TCT GAC CTA TCA GCT TTG GAC GGT-Dig-5'

Reporter: (SEQ ID NO:12)

5'-TAG ATG TGG TAG CCG TTT CTC AGG-FITC-3'

3. Use 100 units of Xho I and Mun I, plus 800 units of Exo III fordigestion.

EXAMPLE 6 Methicillin Resistant Staphylococcus aureus

Methicillin resistant strains of S. aureus were initially isolated soonafter the drug was introduced for clinical use. Resistant strainsproduce a penicillin-binding protein with low affinity for -lactamantibiotics, thus rendering the pathogen resistant. This protein isproduced by an acquired gene, mecA, which is the target for TPAdetection (45). DNA is isolated from bacterial colonies growing onsensitivity disk agar (Nissui) by the method of Cassiday et al (46), ordirectly from blood or serum as described above. The followingmodifications will be used for the TPA procedure listed above for thedrug resistant form of S. aureus:

1. Extract DNA from 30 ml whole blood and resuspend in 0.5 ml water.

2. Use 2 nmoles of each probe at the appropriate step:

TFO: (SEQ ID NO:13)

5'-CCA TTT TTC CCT GAG CTT TTT-3'

Capture: (SEQ ID NO:14)

5'-TAA TTC TTC AGA GTT AAT GGG A-Dig-5'

Reporter: (SEQ ID NO:15)

5'-AAC ATG AAG ATG GCT ATC GTG TC-FITC-3'.

3. Use 100 units of Sal I and Mnl I, plus 800 units of Exo III fordigestion.

EXAMPLE 7

Assay combinations

The following is a listing of combinations of target nucleic acids andprotection molecules. The present invention is not limited to theseexamples and other combinations can be used by those skilled in the art.

    ______________________________________                                        Target Nucleic   Protection                                                   Acid             Molecule                                                     ______________________________________                                        ss DNA           ss DNA                                                                        ss DNA with                                                                   crosslinkers                                                                  ss DNA that covers                                                            restriction sites                                                             with homologous                                                               bases/non-cross                                                               linking                                                                       ss DNA with                                                                   crosslinkers and 5'                                                           non-homologous                                                                tail                                                                          ss RNA with                                                                   crosslinkers                                                                  ss RNA with non-                                                              homologous RNA                                                                tail on either side of                                                        target                                                                        ss RNA with                                                                   crosslinkers                                                                  ss RNA                                                                        ss RNA +                                                                      Antibody to hybrid                                                            ss RNA with non-                                                              complementary                                                                 RNA tail (either                                                              one or both sides of                                                          target sequence).sup.1                                                        ss RNA with non-                                                              complementary                                                                 RNA tail, 3' side                                                             target.sup.1                                                                  ss RNA with non-                                                              complementary                                                                 RNA tail, 5' side                                                             target.sup.1                                                                  ss DNA extending                                                              beyond flanking                                                               regions and                                                                   covering restriction                                                          sites.sup.3                                                                   ss DNA                                                                        homologous to                                                                 target site.sup.3                                                             ss DNA                                                                        complementary to                                                              target                                                                        ss RNA with non-                                                              complementary                                                                 DNA tail (either                                                              one or both sides of                                                          target sequence).sup.1                                                        ss RNA with non-                                                              complementary                                                                 DNA tail, 3' side                                                             target.sup.1                                                                  ss RNA with non-                                                              complementary                                                                 DNA tail, 5' side                                                             target                                                                        ss DNA with non-                                                              complementary                                                                 RNA tail (either                                                              one or both sides of                                                          target sequence)                                                              ss DNA with non-                                                              ss DNA with non-                                                              complementary                                                                 RNA tail, 3' side                                                             target                                                                        RNA tail, 5' side                                                             target                                                                        ss DNA with non-                                                              complementary 5.sup.1                                                         DNA tail                                                                      ss RNA + DNA                                                                  ss RNA with non-                                                              complementary                                                                 RNA tail, 5' side                                                             target.sup.1                                                                  ss DNA target                                                                 specific only with                                                            TFO                                                                           ss DNA with non-                                                              complementary                                                                 5' tail.sup.2                                                                 ss RNA with non-                                                              complementary                                                                 RNA tail on both                                                              sides of target +                                                             antibody to hybrid                                                            ss RNA with non-                                                              complementary                                                                 RNA tail on both                                                              sides of target                                                               ss RNA with non-                                                              complementary                                                                 5' tail.sup.2                                                                 ss RNA with non-                                                              complementary 5'                                                              DNA tail                                                     ds DNA           TFO DNA or RNA                                                                PNA                                                                           PNA with non-                                                                 complementary 5'                                                              DNA tail                                                                      Sequence specific                                                             protein                                                      ss RNA           PNA                                                                           ss DNA +                                                                      Antibody to                                                                   DNA/RNA hybrid                                                                ss RNA.sup.3                                                                  ss DNA.sup.3                                                                  ss RNA with cross                                                             linkers                                                                       ss Dna with cross                                                             linkers                                                      ss RNA           Sequence specific                                                             protein                                                      ds RNA           TFO (RNA)                                                                     TFO (DNA)                                                                     PNA                                                          ______________________________________                                         .sup.1 + Antibody to DNA/RNA hybrid can also be used to aid in binding        .sup.2 binding aided by pH and ionic strength                                 .sup.3 PNA can also be added.                                            

Location of Affinity Molecule

On the ss DNA probe

On ss DNA

On oligo homologous to upstream 5' DNA (target nucleic acid) tail

On oligo homologous to 5' probe (protection probe) tail

On ss RNA probe

On ss RNA

On oligo homologous to 5' DNA (target nucleic acid) tail generated

On oligo homologous to RNA (protection probe) tail

On oligo homologous to RNA tail

On ss RNA cross-linked probe

On antibody

On antibody used in capture system

On antibody to hybrid protection structure

On PNA (peptide-nucleic acid)

On TFO (triplex forming oligo)

On oligo complementary to 5' flanking region of target

On oligo complementary to 5' DNA probe tail

On oligo complementary to 5' flanking region of target

On oligo attached to PNA

On oligo attached to TFO (DNA/RNA)

On sequence specific protein

Location of Reporter Molecule

On ss DNA probe

On ss DNA

On oligo homologous to downstream 5' DNA tail

On oligo homologous to upstream 5' DNA tail

On oligo homologous to generated 5' DNA tail

On oligo homologous to 5' probe tail

On ss RNA

On ss RNA probe

On oligo homologous to RNA tail (probe)

On ss RNA crosslinked probe

On antibody

On antibody used in capture system

On antibody to hybrid protection structure

On PNA (protein-nucleic acid)

On TFO (triplex forming oligo)

On oligo complementary to 5' flanking region of target

On oligo complementary to 5' DNA probe tail

On oligo complementary to 5' flanking region of target

On oligo attached to PNA

On oligo attached to TFO

On sequence specific protein

Antibodies to the hybrid protection structure of DNA/RNA, either DNAtarget with RNA protection probe, or RNA target with DNA protectionprobe, can be used as a capture system.

Peptide Nucleic Acid (PNA) can be used in the second hybridization step.

Triplex Forming oligonucleotides (TFO) can be used in the secondhybridization step.

It should be understood, of course, that the foregoing relates only topreferred embodiments of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 15                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 16 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 # 20               CTGT                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 # 20               AGTA                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 # 20               TTTC                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #                24AAAC GCTG                                                  - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #21                TAAA G                                                     - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #  18              GC                                                         - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #                23GCTC CTC                                                   - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #                24ATAC TGAT                                                  - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #                 22CGG GG                                                    - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #                24TGGA CGGT                                                  - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #                24TTCT CAGG                                                  - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #21                TTTT T                                                     - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #                 22TGG GA                                                    - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #                23TCGT GTC                                                   __________________________________________________________________________

It is claimed:
 1. A method for genetic testing, comprising:a) obtainingisolated unlabeled nucleic acid sequences from a first organismsuspected of containing one or more target nucleic acid sequences thatare capable of indicating a genetic relationship with a second organism;b) contacting at least one target-protecting molecule that specificallybinds to the one or more target nucleic acid sequence with the unlabelednucleic acid sequences of step a) under hybridizing conditionssufficient to form at least one protected target nucleic acid sequence(PNAS); c) digesting the isolated nucleic acids containing at least onePNAS of step b) with enzymes to form at least one PNAS structure havingat least one 5' single-stranded region generated by enzymatic digestion(PNAS/tail); d) hybridizing a reporter probe to the at least onePNAS/tail of step c) wherein the reporter probe binds to asingle-stranded region of the at least one PNAS/tail; e) detecting theat least one PNAS/tail as indicative of the presence of the one or moretarget nucleic acid sequence; and f) determining the geneticrelationship between the first and second organism.
 2. The method ofclaim 1, wherein the first and second organisms are plants, animals,including humans, or microorganisms.
 3. The method of claim 1, furthercomprising:prior to step e) hybridizing a capture probe thatspecifically binds to a single-stranded region of the at least onePNAS/tail.
 4. The method of claim 1, wherein the at least one PNAScomprises a triplex structure.
 5. The method of claim 1 wherein the atleast one PNAS comprises a duplex structure.
 6. The method of claim 1wherein the at least one PNAS comprises a protein.
 7. A method fordetecting organisms in a sample, comprising:a) obtaining isolatedunlabeled nucleic acid sequences from the sample suspected of containingat least one organism; b) contacting at least one target-protectingmolecule that specifically binds to at least one nucleic acid sequenceof the at least one organism with the unlabeled nucleic acid sequencesof step a) under hybridizing conditions sufficient to form at least oneprotected target nucleic acid sequence (PNAS); c) digesting the isolatednucleic acids containing at least of PNAS of step b) with enzymes toform at least one PNAS structure having at least one 5' single-strandedregion generated by enzymatic digestion (PNAS/tail); d) hybridizing areporter probe to the at least one PNAS/tail of step c) wherein thereporter probe specifically binds to a single-stranded region of the atleast one PNAS/tail; and c) detecting the at least one PNAS/tail asindicative of the presence of the at least one organism.
 8. The methodof claim 7, further comprising, hybridizing a capture probe thatspecifically binds to a single-stranded region of the at least onePNAS/tail prior to the step of detecting the at least one PNAS/tail. 9.The method of claim 7, wherein the at least one PNAS comprises a triplexstructure.
 10. The method of claim 7, wherein the at least one PNAScomprises a duplex structure.
 11. The method of claim 7, wherein the atleast one PNAS comprises a protein.
 12. The method of claim 7 whereinthe sample is from an organism or the environment, and is cellular,tissue, or fluid.
 13. The method of claim 12 wherein the sample isdonated blood or organs.
 14. The method of claim 7 wherein at least oneof the pathogenic organisms is capable of causing infectious disease.15. The method of claim 7, wherein the reporter probe of step d) furthercomprises an affinity molecule.
 16. The method of claim 7, whereinsignal from the reporter probe of step d) is amplified.
 17. The methodof claim 8, wherein the reporter probe and the capture probe arehybridized to the at least one PNAS/tail in the same step.
 18. Themethod of claim 1, wherein the reporter probe of step d) furthercomprises an affinity molecule.
 19. The method of claim 1, whereinsignal from the reporter probe of step d) is amplified.
 20. The methodof claim 3, wherein the reporter probe and the capture probe arehybridized to the at least one PNAS/tail in the same step.